C90RF72: Bound to Repeat Itself

The linking of the C9ORF72 repeat expansion mutation in 2011 to ALS caused a great deal of excitement in the research community.

ALS caused by a C9ORF72 repeat expansion appears to be the same — and amenable to the same treatment strategies — in both familial and sporadic cases of the disease.

Related articles Antisense Against C9ORF72 and C9ORF72 Research Models outline the work of researchers who are working to: study the nature of the mutation; discover the mechanism by which the mutation causes ALS; develop cellular and animal models in which to study the disease; and develop therapies for ALS caused by C9ORF72.

In September 2011, two research teams working independently added chromosome 9 open reading frame 72 (C9ORF72) to the list of genes that, when mutated, can cause amyotrophic lateral sclerosis (ALS).

The finding sparked a great deal of interest in the ALS research community for several reasons, including:

its classification as a repeat expansion mutation;

the association of the mutation with another disorder already linked to ALS, frontotemporal dementia (FTD); and

the significant percentage of ALS cases attributable to mutations in the C9ORF72 gene — about 40 percent of familial ALS (where there is more than one occurrence of the disease in the family), and approximately 7 to 10 percent of sporadic ALS (in which there is no known occurrence of the disease in other family members).

(Note: Originally, the sporadic ALS percentage was thought to be around 4 percent.)

Here’s a look at what scientists have discovered about C9ORF72 in ALS and potential therapeutic strategies already under development to counteract it.

Repeat this

The two research teams that uncovered the C9ORF72 mutation were led by neurologist Bryan Traynor, head of the Neuromuscular Diseases Research Unit at the National Institutes of Health in Bethesda, Md.; and Rosa Rademakers, associate professor of molecular science at the Mayo Clinic Florida in Jacksonville.

Traynor’s team conducted studies first in a Finnish population of people with either familial or sporadic ALS, along with members of one ALS-affected family each from the Netherlands and the United Kingdom. The team then studied ALS patients of European descent, including people with familial ALS from Germany, Italy and North America.

Rademakers and colleagues studied a group of people with familial FTD or ALS from Vancouver, B.C., Canada, San Francisco, and Mayo Clinic sites in Florida and Minnesota.

They found that the ALS-associated defect in C9ORF72 belongs to a class of mutations called repeat expansion mutations. A repeat is a sequence of nucleotides (the bases, or building blocks, of DNA designated A, C, G and T) that occurs sequentially multiple times in a particular stretch of DNA. A repeat expansion occurs when an error causes the number of repeated segments to increase.

The chromosome 9-linked ALS mutation is a hexanucleotide repeat expansion in which a six-letter string of nucleotides coded GGGGCC is repeated far more times in people with ALS, FTD or ALS-FTD than in people unaffected by either or both of the two disorders.

Expansion issues

C9ORF72 is the first repeat expansion mutation found to directly cause ALS, although repeat expansions in the ataxin 2 and NIPA1 genes have been identified as significant contributors to the risk of developing the disease. NIPA1 also has been shown to modify the ALS disease course.

The average number of hexanucleotide repeats in people unaffected by ALS appears to be about three, with the maximum number rising only to around 30. Although the exact numbers of repeats observed in individuals with ALS has yet to be determined, a possible range of 700 to 1,600 has been suggested by several investigators.

Because repeat expansion mutations have been implicated in a number of other diseases, they’ve been the subject of a great deal of research — a resource upon which ALS researchers are drawing in their quest to determine how the C9ORF72 mutation causes disease, as well as in their efforts to design therapeutic strategies to counter it.

Characteristics of C9-related ALS

It’s thought by some that the C9ORF72 repeat length may correlate with particular aspects of the disease. For example, a longer expansion might correspond with an earlier age at onset. Until scientists are able to precisely measure the expansion, however, this can’t be confirmed.

What is known is that in studies comparing people with ALS caused by C9 mutations to people with ALS caused by mutations in the SOD1, TDP43 or FUS genes, or resulting from unknown causes, ALS caused by the C9ORF72 repeat expansion mutation:

tends to result in a more severe disease course with rapid progression;

has a higher rate of bulbar onset (where muscle weakness first begins in the mouth and throat);

is more often associated with cognitive impairment and FTD;

often manifests at an older age; and

generally correlates with a shorter survival time.

Prior associations already had been established linking bulbar onset, rapid progression and shorter survival time in ALS. The discovery of C9ORF72 ties them more tightly together and links them to a subset of people with ALS whose disease shares the same molecular cause.

Adriano Chiò

“What is different, on the other hand, is the frequency of the mutation in different populations,” says neurologist Adriano Chiò, professor of neurology at the University of Torino in Turin, Italy. “C9ORF72 seems to be more frequent in people with northern European ancestry, followed by those of southern European ancestry. There also are data indicating that it can be relatively rarer in Asian populations.”

An age-associated disease

Although presence of the C9ORF72 mutation is sufficient to cause ALS, manifestation of the disease is age-dependent.

People under age 25 who have the C9ORF72 mutation are unlikely to show signs of ALS.

The disease manifests in 50 percent of those with C9ORF72 who have reached the age of 58.

In people 80 years old with the C9ORF72 mutation, 99.9 percent will have ALS.

It's likely that a number of genetic influences affect onset and progression of C9ORF72-related ALS.

C9ORF72 in both familial and sporadic ALS

ALS caused by a C9ORF72 repeat expansion appears to be the same — and amenable to the same treatment strategies — in both familial and sporadic cases of the disease.

On a practical level, the implications are clear: Therapies designed for C9ORF72-related ALS should work for anyone with the C9ORF72 expansion mutation.

But on a separate level, it raises a question: In C9ORF72-associated ALS, what exactly distinguishes familial from sporadic?

In a study of a large group of people with sporadic C9ORF72-related ALS, Chiò and colleagues found that “at least one third of these patients were not really sporadic — they had a parent with FTD, but no other cases of ALS in the family.”

One implication of this finding, Chiò says, is that, “We now have to consider FTD in the relatives when we are establishing whether a patient with ALS is ‘sporadic.’”

Chiò says it’s possible that some of the apparent sporadic cases of the disease could be caused by an increase in the size of the expansion in an individual compared to its size in his parents. “This phenomenon is well-known in repeat disease,” he says, “and is called ‘instability’ of the DNA.”

Started in Scandinavia?

Others have a different take on C9ORF72 and sporadic ALS — that it’s really all familial.

"We don't think the C9ORF72 mutation in ALS happens spontaneously," says Philip Wong, professor in the departments of pathology and neuroscience at John Hopkins University School of Medicine in Baltimore.

“Genetics support the idea that it spread from one founder to those who have it today.” A founder in this context is a person in whom a genetic error occurs; the mutation then can be passed down to successive generations.

Traynor says data suggest that the C9ORF72 mutation started in one person in Scandinavia approximately 1,500 years ago. “We can actually trace it around the world based on the movements of the Vikings and their descendants,” he notes.

“Individuals that carry the C9ORF72 mutation and who have apparently sporadic disease share the same haplotype (combination of DNA sequences on a chromosome) as individuals who definitely have familial ALS and who carry the expansion,” Traynor says. “What that tells us is that those sporadic individuals are part of the same family.”

Loss or toxic gain of function?

Based on disease research in other fields — particularly myotonic muscular dystrophy, which also is caused by a repeat expansion mutation — most scientists generally agree that there likely are two possible ways in which C9ORF72 might cause ALS.

One possible mechanism is called toxic gain of function. It involves a new, abnormal function or activity of the protein associated with the flawed gene. For example, the extra genetic material that makes up the repeat expansion could inappropriately bind or “sop up” proteins crucial to the protein-building process during the RNA stage. (RNA is a chemical cousin to DNA and the chemical step between DNA and protein synthesis.) The evidence for a gain-of-function mechanism comes from at least one study, in which scientists found clumps of expanded RNA in brain tissues collected from people who had ALS and carried the expanded C9ORF72 repeats.

The second possible way in which the C9ORF72 mutation may be causing ALS is called loss of function. This describes the loss of the protein that normally would be encoded by C9ORF72 and the resulting loss of whatever task(s) that protein is responsible for doing.

In at least two studies, it’s been shown that expanded repeats in C9ORF72 correlate with reduced activity of RNA. This could suggest at least a partial loss-of-function mechanism.

A number of researchers are working to figure out which of the two hypotheses is the right one — or if, perhaps, it’s a combination of the two. Some are studying the mutation using cell culture models; some are developing research animal models, which they’ll then use to study the disease.

Others are bypassing the question of “how” for the moment, seeking instead to engineer strategies for blocking the C9ORF72 expansion — or even the entire gene. This may allow them to both develop therapies and shed light on the disease mechanism simultaneously.

Test available

In the meantime, Athena Diagnostics has begun offering the first clinically available test designed to detect expansion in the C9ORF72 gene.

The test is offered for physicians to use in helping determine or refine an ALS diagnosis.

Anyone considering the test should talk with their physician. Consultation with a genetic counselor, who can help obtain and interpret the results of genetic testing, is strongly recommended.